biology essay

How Proliferation Can Be Affected Biology Essay

Published: 23, March 2015

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A cell culture grows by the multiplication of cells. The cell cycle occurs to identically copy DNA into two daughter cells. The cell cycle is made up by two basic steps which are interphase and Mitosis. A cell must go through these phases to ensure enough genetic material to produce two identical daughter cells with the same amount of DNA as the mother cell. A cell will spend most of its time in interphase which can be up to 12 hours and much less time in mitosis, about an hour (Alberts et al 2008). Interphase is made up of 3 steps which are G1, S, G2. G1 is when a cell is between mitosis and DNA replication. If there is no signal during G1 to tell the cell to continue the cycle the cell goes into G0. The G0 phase is when a cell leaves the cycle either permanently or when resting and will no longer divide. S is the phase of the cycle which is found between G1 and G2 when DNA is synthesised. The G2 phase is the phase between S and M. The M phase is when mitosis occurs. Mitosis involves four stages which are prophase, metaphase, anaphase and telophase. During prophase the chromatin condenses and the chromosomes become visible under a light microscope as two chromatids joined by the centromere. The mitotic spindle forms and the nuclear envelope disassembles. During metaphase the chromosomes line up along the spindle equator and the sister chromatids attach to opposite poles of the cell. In anaphase the chromosomes split and each sister chromatid is pulled to opposite ends of the cell. The spindle disassembles and the nuclear envelope reforms around the uncoiling chromosomes for telophase. Cytokinesis is the cleaving of the cells to form the two new daughter cells and occurs after mitosis. The cell cycle has now returned to interphase.

A diagram of the cell cycle that is involved in producing daughter cells

Figure 1: The Internet Encyclopedia of Science(No Date)

According to Greenfacts (2009) "Cell culture can be defined as the process of growing cells in laboratory conditions, commonly on a glass surface immersed in nutrient fluid." Eukaryotic cells are difficult to grow as they require complex media such as cDMEM, furthermore they are at risk of contamination by microbes such as yeast, fungi and bacteria. In the media, foetal calf serum(FCS) was used. The serum encourages growth by adding factors such as proteins and hormones and acts as a buffer. The growth of the cultures has to be done in sterile conditions in a laminar flow hood to prevent contamination. The flow hood is sterilized using uv light and a high efficiency particle filter, to provide a continuous displacement of air. To ensure constant sterile conditions nothing could be put over the air flow, the hood was to be kept tidy and wiped down with ethanol before and after use. Also lids were to be put down so that the area exposed to the culture was not touching the surface and any instruments used (such as pipettes and) were sterile and could not touch other objects to ensure that they stayed sterile.

The cell culture which was grown was human prostate cancer cells (PC3 cell lines). The PC3 cell lines were used to show the difference in proliferation, in the presence or absence of the medium cDMEM.To collect data to show the proliferation of the PC3 cells, a number of treatments were necessary before the cells could be viewed. Once the cells were in suspension the culture could be examined using a number of techniques. These included using a haemocytometer, immunocytochemistry, MTT assay and flow cytometry.

A haemocytometer is a special slide used to count cells in suspension. It has an area of known volume which is under a cover slip. The haemocytometer is split into small squares only visible through the microscope. The cells can be counted inside each square and calculated to give a total number and an average number of cells that are present in the suspension. To collect the data, the cells from two of the squares were counted and averaged. (To get a more accurate result more of the squares could have been counted and the average cell number calculated.) Once this was found it could be used to produce dilutions.

A haemocytometer to measure the number of cells

The squares used to count the number of cells present and averaged.

Figure 2: Vineeth(2008)

According to IHC world LLC (2010), "Immunocytochemistry is a technique used to detect the presence of proteins in cell suspension, cultured cells or cytospin through the use of a specific antibody." The cell culture needed to be prepared before they could be tested. There are many ways in which to prepare cells for analysis using immunocytochemisty. To prepare the PC3 culture, it was grown in medium and pre-treated with trypsin, which removed the cells from the flask surface, before being centrifuged and prepared on slides using the cytospin. Acetone/Methanol was used to fix the cells to the slides. Peroxidase blocking buffer was used to stop non-specific staining. The TBS that the slides are washed with is used as a buffer to maintain pH. Normal goat serum acts as a blocking agent to stop non-specific binding of proteins, before mouse anti-human Ki-67 is added. Timke et al (2006) states that, "To assess the tumor cellular proliferation (% Ki-67 positivity), immunohistochemical staining was done using the mouse anti-human Ki-67 antigen."Fernades et al (2010) suggests that, "Ki-67 is expressed across the active phases of the cell cycle and serves a biomarker for cell proliferation."

The MTT method is used to measure cell proliferation. Edmundson et al (1988) states "The assay involves conversion of the yellow tetrazolium salt MTT by viable proliferating cells to an insoluble product, blue formazan." The cells are prepared before adding MTT to the wells. They are firstly incubated in cDMEM medium at the same concentration throughout the wells. New media was made at different concentrations to measure change in growth with different volumes of FCS. After incubation, the MTT was added to the wells and acid-isopropanol was added to allow the blue formazan crystals to dissolve. The wells were then measured at an absorbance of 570nm.

Flow cytometry is used to measure physical and chemical characteristics of cells. It determines the phase of the cycle the cells are in, the size and granularity, shape and components within the cell (Cancer Research UK 2004). The fluorescent light emitted by the machine is passed through the solution to be measured and sets of filters and optics receive the light and produce results in the form of graphs (Cancer research UK 2004).

To find out the how proliferation occurred using PC3 cells, a number of different methods were used. Cell cycle analysis was done using a flow cytometer to find out which cells were in the different phases. The cells were treated with FCS to analyse how this affected cell proliferation using the MTT assay and immunostaining was done to show how well Ki-67 could be used to show proliferation by attaching to specific proteins. The cells were incubated with FCS at different points during the experiment and the results were done to show how this nutrient affected the PC3 cells over a period of growth time.

Method

In the laminar flow hood using sterile pipettes, 100ml of media (cDMEM) was made up using 5ml foetal calf serum (FCS), 1ml penicillin/streptomycin and 43ml DMEM. The supernatant was poured off the PC3 cells, 10ml PBS added and swirled in the flask to wash the cells and then discarded. 2-3ml trypsin EDTA was added and the flask was placed in the CO2 incubator for 2 minutes or until the inverted microscope showed that the cells had detached and rounded up. Media (10ml) was added to the flask and then the suspension was poured into a centrifuge tube (15ml).The cells were centrifuged for 5 minutes at 1000rpm. The supernatant was discarded and the pellet was re-suspended in 3ml cDMEM. 50ï­l was placed on a haemocytometer to count the number of cells.

cDMEM was made up to 15ml from 21.1ï­l of cell suspension. 200ï­l was added to the middle 60 wells of the 96 well plate and PBS to the outer wells. The plate was placed in the incubator for 7 days.

The cells in the 96 well plate were checked under the microscope to ensure growth and FCS was then diluted to 0, 1, 5, 10 and 20% and made up to 6ml cDMEM using serum free media. The supernatant from the wells was disposed of and the wells were washed with PBS, before adding the measured concentrations of media (200ï­l) to 12 wells each.

20%=6ml x 0.2=1.2ml

10%=6ml x 0.1=0.6ml

5%=6ml x 0.05=0.3ml

1%=6ml x 0.01=0.06ml

Table 1: The percentage of dilution of FCS with cDMEM

1

2

3

4

5

6

7

8

9

10

11

12

A

B

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

C

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

D

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

E

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

F

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

G

0%

0%

1%

1%

5%

5%

10%

10%

20%

20%

H

The plate was then put into the CO2 incubator. 20ï­l of MTT solution (5mg/ml) was added to each well and the plate was incubated at 37oC (4 hours). The medium was removed and acid-isopropanol was added to each well (100ï­l). Then the plate was left at room temperature (30 mins). The blue formazan crystals formed were measured at 570nm.

From the cell suspension remaining after the 96 well plate was filled, 283ï­l was used and made up to 20ml in two T75 flasks. After 3 days, the medium was removed and the cells washed with PBS, before fresh cDMEM was added to one flask and serum free medium to the other. The flasks were then incubated further four days.

The medium was removed from the T75 and the cells were washed. Trypsin EDTA was added (2-3ml) and the flasks were incubated for 2-5 minutes. The trypsinised cells were poured into tubes, cDMEM (3ml) was used to wash any remaining cells from the flask into the tubes and the tubes were centrifuged for 5 minutes at 100rpm/400g. The supernatant was removed and the cells were re-suspended in 3ml PBS. The cells were counted using the haemocytometer and diluted using 30ï­l cell suspension and made up to 1ml using PBS.

4ml of ice-cold 80% ethanol was added to the remaining cell suspension, slowly, drop by drop, whilst agitating the cells, and placed it in the freezer. The ethanol fixed cells were centrifuged (400mg /1000rpm 10 mins). The supernatant was removed and the pellet was re-suspended in 1ml propidium iodide solution. This was incubated at 37oC (1 hour). The solution was diluted to fit into the flow cytometer's extremities and was then measured using the flow cytometer.

The diluted cells from the T75 flasks were used for cytospin. One spot on the slide was centrifuged, using 200ï­l of the serum free cell suspension, at 1000rpm for 3 minutes and the second centrifuged using cDMEM. The slides were removed from the cytospin and fixed in acetone/methanol for 5 minutes. The slides were then placed in peroxidase blocking buffer for 15 minutes. The slides were washed in TBS for twice for 5 minutes and excess TBS removed with a tissue. The cells were then covered by normal goat serum (15 mins). The cells on one slide were the incubated in a humid atmosphere with 30ï­l Mouse anti-human ki-67 per spot and a cover slip placed on top (1 hour). The slides were washed in TBS three times. Biotinylated Goat anti-mouse (30ï­l) was added to all spots with cover slips and incubated (30mins). The cells were washed with TBS three times. 30ï­l of Vectostain ABC (Avidin/Biotin Complex) was added to the cells and incubated. The cells were washed again with TBS three times. The slides were stained with DAB (20 mins) which was made up of DAB silver (1 tablet), urea gold(1 tablet) and distilled water. The slides were then washed in running tap water (2 mins). The cells were then counterstained with haemotoxylin (1 min) and then washed with running tap water again (3mins). Glycerol/TBS and cover slips were added to view under the microscope and images were taken of the cells.

Results

Tables 2 and 3: The Number of cells found in two squares of the haemocytometer

13

7

4

15

11

9

9

7

7

7

11

12

6

12

7

6

0

6

8

5

2

4

4

8

0

4

4

10

4

2

4

4 Total=143 Total=69

The haemocytometer squares show how many cells were in each smaller square. The average was taken by adding all the cell counts together and dividing it by the number of squares there are. This was then multiplied by 10000 to give a number which represented how many cells were in 1ml.

Calculation to find a dilution to add to the 69 well plate

Average number of cells =total number of cells in square 1 +total number of cells in square 2

2 squares

= =106

Total number of cells in 1ml=average number of cells X 10000 to give number of cells in 1 ml

=106 X 10000=1060000

1500 cells needed=22500 cells needed

number of cells in 1ml

= =0.021ml=21.1ï­l

The calculations above show the averages from the haemocytometer squares and were used to dilute the culture to contain only 1500 cells.

Calculation to find a dilution to add to T75 flasks

300000 cells needed=300000 cells needed

number of cells in 1ml

= =0.283ml=283ï­l

This calculation shows the volume required to dilute the cells using cDMEM to make up a solution of 300000 cells.

Table 4: The haemocytometer cell count for the serum free culture

37

46

Average= x 16x104 =6640000per ml

200000 cells needed= =0.030ml=30ï­l

The volume calculated above (table 4) and below (table 5), using the haemocytometer, is used to make up a solution of 200000 cells made up to 1ml using PBS

Table 5: The haemocytometer cell count for the flask containing cDMEM

22

25

Average=x 16x104=3760000 per ml

200000 cells needed= =0.053ml=53ï­l

The cells were counted on the haemocytometer. Only two squares were counted and multiplied by the number of small squares within the larger square. This was done for both the cDMEM sample and the serum free sample.

Figure 3: Image of cells incubated in cDMEM

The image above shows the cells present in the T75 flasks before the cells were placed on slides by cytospin. This picture is of the cells that were incubated with nutrients in cDMEM.

Figure 4: Image of cells incubated in serum free medium

This image shows the cells that were incubated in T75 flasks with serum free medium. These cells were incubated for the same length of time as the culture in cDMEM but the serum free cells did not have nutrients included in the medium.

Table 6: Number of cells on cytospin slides

Table 6 shows the number of cells counted on each spot of cells on the cytospin slides. There were two different staining of cells on the slides. There were purple stained cells and cells in which the nucleus was stained black. The table shows that purple staining was found on both slides but only the slide with primary antibody showed black staining.

Figure 5: Cells in cDMEM - without Primary Antibody

After Immunocytochemistry, the image above was taken, showing the number of cells on the slide. The cells were cultured in cDMEM which has nutrients for growth included. These cells did not have primary antibodies added and were stained purple.

Figure 6: Cells in Serum Free Medium without Primary Antibody

The image above shows the cells which have been growth without nutrients in serum free medium. They haven't had primary antibody added to them and the slides were viewed under a microscope after being treated for immunocytochemistry. The cells on this spot show only purple staining.

Figure 7: Cells in cDMEM with Primary Antibody

The cells in the picture above were treated with primary antibody and were incubated with cDMEM. As seen, the cells in this culture show purple staining and some with the nucleus stained black.

Figure 8: Cells in Serum Free Medium with Primary Antibody

The cells in the culture above show some black staining of the nuclei and also purple staining. These cells were incubated in serum free medium and treated with primary antibody.

Table 7: Absorbances of the Blue Formazan Crystals in the MTT 96 Well Plate in different concentrations

PBS

0%

1%

5%

10%

20%

Table 7 shows the absorbances that are taken after the cells have been treated with acid-isopropanol to remove the blue formazan crystals, which have been formed by adding MTT solution. A higher absorbance shows cell apoptosis and proliferation rate. The values of absorbance increased as the concentration of FCS increases.

Table 8: Average Absorbances from each Lane of 96 Well Plate

The averages above were calculated from cells in 6 wells each. The first and last lanes contained PBS. The first two lanes after PBS were filled with 0% FCS media. The second two were filled with 1% FCS, lanes 6 and 7 had 5%, 8 and 9 were filled with 10% and lanes 10 and 11 were filled with 20% FCS. The results show an increase in absorbance. There are a few lanes which show anomalous results and these were included in this table and the graph below, however, the average absorbance was recalculated later without including the outliers.

Figure 9: The graph shows the average absorbencies for each of the 12 lanes on a 96 well plate

Figure 9 shows that there is an increase in average absorbance across the well lanes until lane 8 where it rapidly falls. The graph then rises again but not to the height from which it fell.

The table shows the absorbance of the cells in the 96 well plate. The green highlighted squares in the table show the outliers which have been found using the mean and standard deviation. The highlighted squares fall outside the mean, plus or minus the standard deviation. The most outliers were found to be in the 10% concentration wells.

All of the answers using the calculations are found not including outliers.

Figure 10: The Average Absorbance of Blue Formazan in Cells at Different Concentrations of FCS Excluding Anomolous Values

The graph above (figure 10) shows the mean results for the absorbance found at each concentration of FCS. In this histogram the mean has been calculated without the outliers. The bars show that the absorbance increases until 5% concentration and the 10% and 20% bars fall.

Figure 11: The Standard Errors of the Average Absorbance

Figure 11 shows the average absorbance without outliers for the MTT assay. The standard error bars are to show the mean distributed between the number of values. The results show that the absorbance values increases up to 5% and then falls. The standard errors appear at the top of each bar and increase with the absorbance.

Table 10: Results of the T-test using a normal distribustion

20%

0.000042

10%

0.002516

5%

0.000023

1%

0.000353

0%

1.000000

The table shows that all but 0% are significant as they all lie below 0.05.

Table 11: The Number and Percentage of Cells Stained on Each Spot on the Slides.

The table shows the number of cells that have been stained purple and the number of cells which have been stained black. The percentage shows the number of cells in comparison with each other. There are no cells stained black in the cells that were not treated with antibody. The cells that were incubated in cDMEM show more cells that have been stained black than those in serum free medium.

Figure 12: The Number of Cells that showed Staining on each Spot on the Slides

The graph (Figure 12) shows the number of cells on each spot on the slides. The purple lines represent how many cells have been stained purple and the black lines show the black staining of cells. There are no cells stained black without the primary antibody but the cells that were treated show many cells stained black.

Figures 13 and 14 (above) show that in the serum free medium, the cells are smaller than in serum and that the cells are of similar granularity in both.

Figure 15: Number of Cells in each Stage of Figure 16: Number of Cells in each Stage of

the Cell Cycle for Serum Free Medium the Cell Cycle for Serum Medium

Figures 15and 16 show the number of cells that are in each stage of the cell cycle. M1 shows cells that are in G0 and G1. M2 shows cells that are going through synthesis and M3 shows cells that are in G2 and Mitosis stages. The graph for serum free has more cells in M1 as it has a higher peak than medium with serum. The serum medium shows more cells in M3 than in serum free medium.

Total number of cells in serum free=M1 + M2 + M3

=78.34+7.32+11.92=97.58

Number of live cells in M1= M1

in serum free Total number

= 78.34 x100

97.58

=80%

Number of live cells in M2= M2

in serum free Total number

= 7.32 x100

97.58

=8%

Number of live cells in M3= M1

in serum free Total number

= 11.92 x100

97.58

=12%

Total number of cells in serum = M1 + M2 + M3

=71.99+7.86+16.81

=96.66

Number of live cells in M1 in serum= M1

Total number

= 71.99 x100

96.66

=74%

Number of live cells in M2 in serum= M1

Total number

= 7.86 x100

96.66

=8%

Number of live cells in M3 in serum= M1

Total number

= 16.81 x100

96.66

=17%

The calculations above were done to find the percentage of live cells in each phase for each medium the cells have been cultured in.

Table 12: Number of cells in each stage of the cell cycle

Control

FCS

M1(G0, G1)

78.34

71.99

M2(S)

7.32

7.86

M3(G2,M)

11.92

16.81

There are more cells which are in the M1 phase in the serum free medium that the medium containing FCS but there are more cells in M2 and M3 in the medium containing FCS and this can be seen in the table above.

Figure 17: Number of Cells in Each Phase of the Cell Cycle

In this graph the greatest numbers of cells are in M1 phase of the cell cycle. There are a higher number of % gated cells in M1 for serum free medium and more cells in M2 and M3 for FCS containing medium.

Discussion

The data recorded from the experiments confirm that the cultures incubated in serum showed the most proliferation. Most of the results prove that the FCS caused a greater proliferation. The cell growth increases in the presence of cDMEM as the medium includes nutrients. The serum free medium shows a lower cell growth and this is shown by the MTT assay, as the absorbance increases as the percentage volume of FCS increases. At 5% concentration of FCS the growth is at its highest and then the 10% and 20% concentration the absorbance falls. This fall is not a large one which may suggest errors in the absorbance values due to experimental errors, such as re-suspension of the blue formazan crystals. On the flow cytometer the results show that most cells are in M1 which is expected, as most cells spend most of their life cycle in this stage ready to enter mitosis. The flow cytometer showed that a larger proportion of cells in serum were in the mitosis phase than the serum free cells, due to the nutrients, enabling better growth conditions. There is more black staining in the cells with primary antibody added and a higher number in the medium with cDMEM.

The haemocytometer shows that in the serum free there were a higher number of cells present than in the culture grown in cDMEM. The results show this because only 2 squares in the larger square were counted. If more values had been found and averaged I think the haemocytometer would show more cells in the serum sample. I believe this is the case as the images taken on the microscope show that there are more cells in the culture incubated in serum (figure 3)than the control (serum free)(figure 4).

The images taken after the immunocytochemistry was completed show that the sample that was incubated with serum but wasn't treated with the primary antibody (mouse anti human Ki-67)(figure 5) had many cells but only showed purple staining. The nuclei of the cells don't show black staining. This is because the substances (as described in the method) could not bind to the normal goat serum the same as the one which had been treated with the antibody. In the presence of Ki-67, the cells show black colouration. The next picture (figure 6) shows the cells that have been cultured in serum free solution without the primary antibody. These show no black staining and just purple stained cells. Figure 7 showing cells which have been treated with primary antibodies and have been incubated with serum free solution shows cells with black staining and purple staining. There are fewer cells with black staining than purple staining on this microscope slide spot. This is because only some of the cells took up the primary antibody. This may be because the protein that is needed to retain the primary antibody is not present. The last image (figure 8) was taken of cells incubated in serum cDMEM where primary antibody binding has occurred. In this picture there are the same numbers of cells with black staining as purple staining. This is because the cells have been incubated with cDMEM, so the cells have been provided with nutrients. The cells grown without serum have fewer cells with black staining.

The results for immunocytochemisty had a large number of black staining in the cells which had been treated with primary antibody. The numbers of cells with purple and black staining were counted and calculated as percentages (figure 11). The cells which had primary antibody added showed cells which had been stained black and purple, and the cells without primary antibody, showed only purple staining. This means that the cells which had primary antibody on them, were able to take up the Ki-67 into the cells and a complex was made using solutions in the next steps, which were added to the cells as described in the method. Sheffield Hallam University(NO DATE ) states that "The presence of Ki-67 is indicated by a black/brown colouration in some cells." The cells which took up the mouse anti-human Ki-67 were able to show this black staining on the cytospin slides. Ki-67 is used as a marker in cells. Ki-67 stood out from other antibodies produced, because it only reacted with cells which were proliferating in cancer cells. Ross and Hall(1995)

The MTT assay gave absorbances given in table 9. These show a pattern of increasing absorbance as the concentration increases. This is because they are being exposed to higher concentrations of FCS and are therefore getting more nutrients from the medium. This table doesn't highlight any outliers. The average absorbance increases as seen on figure 10 due to the increased amount of nutrients available to the cells. When the graph reaches lane 8 on the 96 well plate it falls. This is due to outliers. This may be because of a large amount of cell death or a small volume of blue formazan crystals being produced in the cell. The larger anomalous results in the tables and graphs could be due to the blue formazan crystals not being fully suspended in solution before the absorbances were measured. Table 9 showing where the outliers were in the 96 well plate also shows the mean, standard deviation, coefficient of variance and standard error. The results of these illustrate an increase in absorbance and this is also displayed in figure 11 with the bars showing the mean and the lines on the bars showing standard error. The statistics follow the same pattern as the absorbances in the table. The Ttest using normal distribution shows that all the concentrations have an effect on cell proliferation apart from 0%. This is shown by the values all being under 0.05, which shows that they have a significant effect.

Using the flow cytometer, the numbers of cells in M1, M2 and M3 were taken from the graphs and were used to compare the cells which had been treated with primary antibody with those which hadn't. Also those which had been incubated in cDMEM and those incubated in serum free medium. The graphs (figures 15 and 16) showing M1, M2 and M3 show that the cells in serum grow better because there are more cells which are in mitosis phase. The percentage of cells in each phase show that there are more cells in serum in the M3 phase than in serum free. There is a very similar percentage of cells in M2 in both and a greater percentage of live cells in M1 in serum free than serum. The graphs showing size and granularity indicates that the cells grow better in medium containing nutrients, cDMEM, because they show that the size of cells in medium are larger than those in serum free medium. The cells size would be bigger in serum because the cells in mitosis are larger as they are dividing. The cells would show a greater granularity because there is double the amount of contents in the cell due to mitosis. This is what would be expected from cells which are in mitosis.

Using the haemocytometer had weaknesses because there were many cells to count within the squares so less squares were counted meaning that the average was less accurate. When counting, it was hard to remember which cells had been counted already and therefore it might have been the case that some cells were missed or cells were counted twice. To combat this problem the mediums could have been diluted and more squares counted, to calculate a more accurate answer. The MTT assay also had a weakness because after waiting 30 minutes for the blue formazan crystals to dissolve from the bottom of the wells, they might not have all dissolved, which means that the absorbance would not have been very accurate.

Other methods can be used to measure cell proliferation using PC3 cells. Rezaiemaneh A, et al (2010) demonstates the use of the MTT assay and flow cytometry to test the effects of anti-EGFR monoclonal antibody on PC3 cells. They then further tested the effects using RT-PCR analysis. Neuwirt et al (2008) uses the flow cytometer and (3)H-thymidine assay to determine the effect of Oligomeric proanthocyanidin complexes (OPC) on PC3 cells.

Generally, from the methods used the PC3 cells showed that when they were in the presence of FCS, they proliferated better than in the serum free medium. The MTT assay, immunocytochemisty and flow cytometer showed positive results in terms of how the cells reacted to FCS, but the haemocytometer showed negative results. This could be due to the weaknesses which were associated with the method.

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